Liver tumor marker sequences

ABSTRACT

Polypeptides whose expression is upregulated in liver tumor cells and cells from liver preneoplastic foci relative to expression in normal liver cells are disclosed as are polynucleotides that encode the polypeptides. In humans, the polynucleotide maps to a region of chromosome 15. The overexpression has also been confirmed in human liver, breast, colon and kidney cancer cell lines. It is believed that the polypeptides are overexpressed in tumor and preneoplastic cells in general.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/620,532 filed on Jul. 16, 2003, which claims the benefit of U.S.provisional application Ser. No. 60/396,626, filed on Jul. 17, 2002.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. Government Support from the followingagency: NIH, Grant No. CA22484. The U.S. Government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

Primary liver cancer is the fifth most common cancer worldwide withapproximately half a million cases reported in 1990. Hepatocellularcarcinoma (HCC) accounts for 80% of all liver cancer and the rates ofHCC have increased by over 70% in the last two decades in the U.S. Thefatality ratio (mortality/incidence) of liver cancer is approximately 1,indicating that the majority of patients live less than a year. Latediagnosis due to lack of clinical symptoms is one of the main reasonsfor the high fatality ratio.

Liver cancer can result from both viral infection and chemical exposure.Known risk factors include hepatitis B and C virus infection andexposure to aflatoxin β1. It is not known whether distinct routes toliver cancer affect the same or different cellular pathways. Nomutational model has yet been developed for liver cancer as it has beenfor other cancers such as colon cancer. The molecular events thatprecede neoplastic transformation of the liver are not well understood.With no clearly identified cause, successful treatment options arelacking. In fact, the specific genes that are deregulated in livercancer have not yet been enumerated. This is a critical first step indeveloping a successful strategy for treating liver cancer.

There is a pressing need to understand the molecular events associatedwith the development of liver cancer, both in humans and in animal modelsystems where liver cancer is extensively studied, and to providediagnostic and therapeutic reagents for treating same.

BRIEF SUMMARY OF THE INVENTION

The invention is summarized in that polypeptides of the invention arefound in liver tumor cells and in cells from preneoplastic liver foci inhuman and non-human animals at levels higher than are found inregenerating or quiescent normal liver tissue. This finding has beenconfirmed in human breast, colon and kidney cancer cell lines. As aresult of this differential overexpression, the polypeptides, as wellsas polynucleotides that encode the polypeptides, are diagnostic markersfor cancer in general, especially liver, breast, colon and kidneycancer, in a human or non-human animal.

In one aspect, the present invention relates to an isolated polypeptidecontaining an amino acid sequence of SEQ ID NO:2, an amino acid sequencethat is at least 70% identical to SEQ ID NO:2 over the length of SEQ IDNO:2, an amino acid sequence of amino acid 22 to amino acid 439 of SEQID NO:2 (secreted portion of SEQ ID NO:2), an amino acid sequence thatis at least about 68% identical to amino acid 22 to amino acid 439 ofSEQ ID NO:2 over the length of amino acid 22 to amino acid 439 of SEQ IDNO:2, an amino acid sequence of SEQ ID NO:4, an amino acid sequence thatis at least 70% identical to SEQ ID NO:4 over the length of SEQ ID NO:4,an amino acid sequence of amino acid 22 to amino acid 400 of SEQ ID NO:4(secreted portion of SEQ ID NO:4), an amino acid sequence that is atleast about 68% identical to amino acid 22 to amino acid 400 of SEQ IDNO:4 over the length of amino acid 22 to amino acid 400 of SEQ ID NO:4.The percentage identity of sequences is determined using the Blosum62alignment method.

In another aspect, the invention also relates to an isolated nucleicacid containing a polynucleotide that encodes a polypeptide of theinvention, to a complement of the polynucleotide, or to a polynucleotidethat is at least about 80% identical, more preferably 90% identical, andstill more preferably 95% identical to an aforementioned polynucleotideof the invention, using the Wilbur-Lipman DNA Alignment method. Apolynucleotide that encodes a polypeptide of the invention can includebut is not limited to SEQ ID NO:1 from nucleotide 25 to nucleotide 1341,which encodes SEQ ID NO:2, as well as SEQ ID NO:3 from nucleotide I tonucleotide 1200, which encodes SEQ ID NO:4. SEQ ID NO:3, predicted bythe inventors to represent a coding region on human chromosome 15(contig Hs15_(—)10351), is 82.4% identical to the polypeptide-encodingportion of SEQ ID NO:1 using the Wilbur-Lipman DNA Alignment method.

In another aspect, a polynucleotide of the invention is engineered intoa genetic construct downstream from a heterologous promoter not nativelyupstream of the polynucleotide that directs transcription of thepolynucleotide. The genetic construct is introduced into a host cellthat supports transcription of the polynucleotide and translation of theencoded polypeptide which can then be purified using methods known tothose skilled in the art. Alternatively, the construct comprising apolynucleotide of the invention is provided in an in vitrotranscription/translation system for producing the encoded polypeptide.

In yet another aspect, the present invention provides a host celltransfected with a genetic construct of the invention.

In still another aspect, the invention is an antibody that specificallybinds to a polypeptide of the invention.

In yet another aspect, the invention is a method for identifying anagent that modulates the expression of a polypeptide of the invention(e.g., an inducer or suppressor). The method includes the steps ofexposing a cell that contains a polynucleotide of the invention underthe control of its native promoter, measuring the expression of thepolynucleotide in the cell, and comparing the expression to that in acontrol cell that is not exposed to the test agent. A higher or lowerthan the expression in the control cell indicates that the agent canmodulate the expression of the polynucleotide. The expression can bemeasured and compared at either the mRNA level or the protein level.Preferably, a liver, breast, colon or kidney cell (cancerous or normal)is used in the method. More preferably, a human or murine liver, breast,colon or kidney cell is used.

In still another aspect, the present invention is a method of diagnosingcancer or preneoplastic development in a tissue or organ of a human ornon-human animal by measuring the expression of a polypeptide of theinvention in cells of the tissue or organ obtained from a regionsuspected of cancer or preneoplastic development, and comparing theexpression to a normal standard, wherein a higher than normal expressionindicates cancer or preneoplastic development in the suspected region. Askilled artisan can readily establish a normal standard. For example, itcan be the expression level in normal cells of the same tissue or organin the same animal, or it can be an expression level range establishedby testing normal cells of the same tissue or organ of other animals ofthe same species. The expression can be measured and compared at eitherthe mRNA level or protein level.

In a related aspect, the present invention is a method for identifying acandidate human or non-human animal for further cancer screening, wherethe method includes, in one embodiment, the step of determining thelevel of a polypeptide of the invention in a blood or blood-derivedsample from the animal, whereby the animal is identified as a candidatefor further cancer screening when the level exceeds either a normalrange established by the same animal during a period that is tumor-freein the tissue or organ, or a normal range established by other animalsof the same species that are tumor-free in the tissue or organ. Inanother embodiment, the method takes advantage of the expected secretionof the polypeptide and the development of antibodies to the polypeptidein a human or non-human animal that overexpresses the polypeptide in thecancerous or preneoplastic tissue or organ. The method includes the stepof determining the level of an antibody to the polypeptide in a blood orblood-derived sample from the animal, whereby the animal is identifiedas a candidate for further cancer screening when the antibody levelexceeds either a normal range established by the same animal during aperiod that is tumor-free in the tissue or organ, or a normal rangeestablished by other animals of the same species that are tumor-free inthe tissue or organ. It is understood that individuals free of cancer orpreneoplastic development in the tissue or organ may not develop anantibody to the polypeptide. Thus, the normal range for the level of theantibody can be zero.

In still another aspect, the invention relates to a kit suitable for usein a method for determining the level of a polypeptide or polynucleotideof the invention, where the kit contains at least one of an antibodyspecifically directed to an epitope on a polypeptide of the inventionand a polynucleotide that hybridizes to a polynucleotide of theinvention, as well as at least one control sample component for whichthe relative or absolute amount of the polynucleotide or polypeptide ofthe present invention is known, the control sample component beingselected from liver cancer cells, preneoplastic liver cells, normalliver cells, breast cancer cells, normal breast cells, colon cancercells, normal colon cells, kidney cancer cells, normal kidney cells, anextract of any of the foregoing cells, a blood sample from a human ornon-human animal, and a blood-derived sample from a human or non-humananimal.

It is an object of the present invention to provide a polynucleotide anda polypeptide that are differentially expressed in preneoplastic orcancer cells and normal regenerating or quiescent cells in a tissue ororgan of a human and non-human animal.

Other objects, features and advantages of the present invention willbecome apparent upon consideration of the following detailed descriptionof the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the cloning of CRG-L2. a) RT-PCR analysis of CRG-L2 inmouse liver tissues. Because the C3H/HeJ mice used in these studies areinbred, all untreated mice are genetically identical. Accordingly, wehave never observed any differences in CRG-L2 expression in comparisonof individual normal mice. Therefore, quiescent, regenerating, andnewborn RNA samples were prepared from several mice and then pooled sothat the same pooled RNA samples could be used in multiple experiments.Quiescent and regenerating samples are a combination of four livers andnewborn samples are a combination of eight livers. However, it is knownthat tumors display heterogeneous genetic and molecular profiles.Therefore, to take into consideration these possible differences, thetumor samples used in our experiments are from individual mice. b) mRNAstructure of CRG-L2. Alignment of the 5′ and 3′ RACE products suggestthat CRG-L2 mRNA can contain one of three alternative 3′UTRs. c)Northern blot hybridization of CRG-L2 in quiescent liver and fourindividual liver tumors. Four bands were detected at 2.4, 3.0, 5.5, and10 kb mRNAs. The three smaller mRNAs correspond to clones A, B, and C. Afourth band, D, was not cloned probably due to inefficient PCR through along 3′ UTR. d) The CRG-L2 open reading frame was aligned to mousechromosome 9 (31 cM). Exons are represented by black boxes. The distancebetween some of the exons is estimated since there are gaps between thecontigs in the genome and these gaps are represented by a >sign. CRG-L2is localized within chromosome 15q21.2 of the human genome and a similarintron/exon structure is suggested by comparing the mouse cDNA to thehuman genome.

FIG. 2 shows structural features of the CRG-L2 protein. a) CRG-L2 cDNAencodes a protein of 48 kD containing two collagen domains and anolfactomedin domain. b) Hydrophobicity analysis of the putative CRG-L2protein with the Kyle-Doolittle algorithm. Positive values representhydrophilic regions and negative values represent hydrophobic regions.

FIG. 3 shows that CRG-L2 expression is increased in human hepatocellularcarcinomas. Top panel is a phosphoimage of the RT-PCR results measuringCRG-L2 mRNA and middle panel is a longer autoradiographic exposure.Equal loading was confirmed by analysis of GAPDH mRNA. All HCC wereclassified as moderately differentiated.

FIG. 4 shows that CRG-L2 expression is restricted in normal tissues.CRG-L2 mRNA was amplified in multiple mouse (a) and human (b) tissuesusing Multiple Tissue cDNA Panels. Aliquots of the PCR products weretaken out at the indicated cycles.

DETAILED DESCRIPTION OF THE INVENTION

Liver cancer is generally studied in animal model systems, preferably inrodent systems, where certain strains are bred for high susceptibilityto liver tumors. C3H/HeJ mice are highly susceptible to liver tumorsafter induction with diethylnitrosamine (DEN). To identifypolynucleotide sequences or genes that show differential expression inliver tumor cells as compared to normal liver cells, gene expressiondifferences between liver tumors and a regenerating liver weredetermined using representational difference analysis (RDA: Lisitsyn, etal., Science 259:946 (1993), incorporated by reference as if set forthherein in its entirety).

In this application, the applicants disclose polypeptides from murineanimals (SEQ ID NO:2) and humans (SEQ ID NO:4) that are upregulated incells and cell extracts from human and murine liver tumors and liverpreneoplastic tissues, relative to quiescent and regenerating normalliver cells. The polypeptide is therefore given the name human or murineCancer Related Gene-Liver 2 (CRG-L2). CRG-L2 overexpression was alsofound in human liver, breast, colon and kidney cancer cell lines. Thus,despite of its name indicative of liver origin, it is believed to beoverexpressed in other types of cancer and preneoplastic cells ingeneral, especially breast, colon and kidney cancer and preneoplasticcells.

Using the Blosum62 alignment method, the human and murine CRG-L2s arefound to be 76% identical. It is expected that CRG-L2s from otheranimals, e.g., other mammals, are at least 70% identical to either thehuman or murine CRG-L2 if compared using the same alignment method.Hydrophobic sequences are present within the first 30 amino acids of SEQID NO:2 and SEQ ID NO:4. Based on information obtained from otherproteins with leader sequences, the serine at amino acid position 21 ofboth SEQ ID NO:2 and SEQ ID NO:4 is believed to be the cleavage site ofa leader sequence for the secretion of both of the CRG-L2s. Accordingly,it is believed that when amino acids 1-21 are cleaved, the remainingamino acid sequences of SEQ ID NO:2 and SEQ ID NO:4 can be secreted fromcells. Corresponding leader sequences on other CRG-L2s can be readilyidentified by a skilled artisan. Depending on the variability of theleader sequences among CRG-L2s, the percentage of identity among thesecreted sequences may be about 3% higher or lower than the overall 70%identity. Generally speaking, it is expected that the secreted portionof CRG-L2s in other animals, e.g., other mammalians, are at least about68% identical to either the secreted portion of the human or that of themurine CRG-L2.

Also disclosed are polynucleotides that encode the polypeptides of theinvention (e.g., the full length and the secreted CRG-L2s), which caninclude, without limitation, mRNA, single or double stranded DNA, cDNAand the like. In addition to the primary murine cDNA product disclosedas SEQ ID NO:1, two additional variant murine cDNAs that are believed toderive from alternative 3′ untranslated regions were also obtained. Thevariant murine cDNA molecules differ from SEQ ID NO:1 in the 3′untranslated portion of the molecules, commencing respectively atnucleotide 1937 and at nucleotide 2342, as shown in the SequenceListing. SEQ ID NO:3 discloses a sequence from human Chromosome 15 thatencodes the human CRG-L2 of SEQ ID NO:4.

Further, the invention provides materials and methods for detectingexpression (and changes in expression) of the polypeptides and of thepolynucleotides that encode the polypeptides, thereby facilitating useas a diagnostic marker for cancer and preneoplastic development and as asystem for assessing putative therapeutic agents. As described in detailin the example below, since the CRG-L2 either belongs or is similar tothe family of cancer-testis antigens, it is expected that a patient willdisplay an immune response to CRG-L2 when it is overexpressed inpreneoplastic and cancerous tissues. Therefore, detecting or measuringthe level of an antibody to CRG-L2 in a blood or blood-derived samplefrom a patient provides another diagnostic tool.

Structurally, the murine CRG-L2 protein (SEQ ID NO:2) contains 439 aminoacids and has a predicted molecular weight of about 47.5 kDA. Using theSimple Modular Architecture Research Tool (available on the world wideweb courtesy of the European Molecular Biology Laboratory—Heidelberg),it was determined that the murine CRG-L2 includes two collagen domainsin the 5′ region (corresponding to amino acids 29-88 and 89-149 of SEQID NO:2, respectively) and a large olfactomedin domain near theC-terminus (corresponding to amino acids 189-433 of SEQ ID NO:2). Thehuman protein also contains two putative collagen domains and oneolfactomedin domain at amino acids 27-85, 86-145, and 177-395 of SEQ IDNO:4, respectively. Olfactomedin-related proteins are secretedglycoproteins having conserved C terminal motifs. It is anticipated thatCRG-L2 can be secreted into the blood and an increase in blood CRG-L2level over normal levels is diagnostic of cancer and preneoplasticdevelopment. Preferably, the diagnostic blood CRG-L2 level is set to beat least about 5%, more preferably at least about 10%, and mostpreferably at least about 25% over a normal level.

The term “isolated nucleic acid” or “isolated polypeptide” used in thespecification and claims of the present invention means a nucleic acidor polypeptide isolated from its natural environment or prepared usingsynthetic methods such as those known to one of ordinary skill in theart. Complete purification is not required in either case. Amino acidand nucleotide sequences that flank a polypeptide or polynucleotide thatoccurs in nature, respectively, can but need not be absent from theisolated form. The polypeptides and nucleic acids of the invention canbe isolated and purified from normally associated material inconventional ways such that in the purified preparation the polypeptideor nucleic acid is the predominant species in the preparation. At thevery least, the degree of purification is such that the extraneousmaterial in the preparation does not interfere with use of thepolypeptide or nucleic acid of the invention in the manner disclosedherein. The polypeptide or nucleic acid is preferably at least about 85%pure, more preferably at least about 95% pure and most preferably atleast about 99% pure.

Further, an isolated nucleic acid has a structure that is not identicalto that of any naturally occurring polynucleotide or to that of anyfragment of a naturally occurring genomic polynucleotide spanning morethan three separate genes. An isolated nucleic acid also includes,without limitation, (a) a polynucleotide having a sequence of anaturally occurring genomic or extrachromosomal nucleic acid moleculebut which is not flanked by the coding sequences that flank the sequencein its natural position; (b) a polynucleotide incorporated into a vectoror into a prokaryote or eukaryote genome such that the resultingmolecule is not identical to any naturally occurring vector or genomicDNA; (c) a separate molecule such as a cDNA, a genomic fragment, afragment produced by polymerase chain reaction (PCR), or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybrid gene, i.e., a gene encoding a fusion protein. Specificallyexcluded from this definition are polynucleotides present in mixtures ofclones, e.g., as these occur in a DNA library such as a cDNA or genomicDNA library. An isolated nucleic acid can be modified or unmodified DNAor RNA, whether fully or partially single-stranded or double-stranded oreven triple-stranded. A nucleic acid can be chemically or enzymaticallymodified and can include so-called non-standard bases such as inosine.

The nucleotide sequences of the invention can be introduced into, andexpressed in, host cells which can be prokaryotic (such as bacterial)cells or eukaryotic (such as yeast, insect, amphibian or mammalian)cells whereupon the transcription of polynucleotide and the propertiesof the encoded polypeptides can be assessed.

The disclosure of the CRG-L2 sequences that are upregulated in livertumor and preneoplastic cells, and in human breast, colon and kidneycancer cell lines provides a means for identifying (in vivo or in vitro)candidates for further testing as preventive and therapeutic agents. Forexample, animal cells that contain a CRG-L2 sequence under the controlof its native promoter can be exposed to a test agent and the effect ofthe test agent on the CRG-L2's expression at the mRNA or protein levelrelative to that of untreated controls can be measured. Alternatively,the level of expression can be assessed in biological samples takendirectly from a human or non-human tissue. Presumably, an anti-tumoragent can bring down the mRNA and protein level in tumor cells.Accordingly, an agent that demonstrates such an activity is a goodcandidate for further testing for anti-tumor efficacy.

The presence and level of such a differentially expressed protein can bereadily discerned using antibodies directed to an epitope on the proteinusing well known methods, such as an ELISA method. It is well within theskill of one of ordinary skill in the art to generate such antibodies.The presence and level of mRNA for the protein can be measured usingmethods for hybridizing nucleic acids (including, without limitation,RNA, DNA, and cDNA). Such methods are generally known to those skilledin the art, but are enabled by the disclosure herein of a tumor-specificsequence. Examples of such methods include but are not limited to RT-PCRamplification, Northern blot and Southern blot.

Given the disclosure herein of polynucleotides that encode CRG-L2 ofhuman, murine and other animal species, one of ordinary skill in the artknows how to design primers for use in RT-PCR analysis and probes forNorthern and Southern blot. The Example below describes a method ofusing RT-PCR to measure CRG-L2 mRNA level in liver tumor cells, liverpreneoplastic cells and normal liver cells. The RT-PCR amplified afragment of CRG-L2 cDNA (SEQ ID NO:1) and its noted 3′ end variants, andthe mRNA level in liver tumor and preneoplastic cells was observed to behigher than that in normal liver cells. Accordingly, a suitable CRG-L2sequence for amplifying or probing in analyzing differential CRG-L2 mRNAlevels is one that corresponds to a fragment shared by all three CRG-L2cDNA sequences. A CRG-L2 mRNA sequence that corresponds to a fragmentunique to the longer 3′ untranslated sequence variants could also beused to analyze differential CRG-L2 mRNA expression since Northernanalysis has shown that all three mRNAs are differentially expressed inliver tumor and preneoplastic cells relative to normal liver cells.

A skilled artisan understands that the polynucleotides disclosed hereincan contain additional nucleotides at the 5′-end, 3′-end or both that donot affect the function of the polynucleotides in terms of their usescontemplated herein. The additional nucleotides can but do not have toassist in the cloning, detection and purification procedures associatedwith the use of the polynucleotides. Similarly, a skilled artisanunderstands that the polypeptides disclosed herein can containadditional amino acid sequences at the N- or C-terminus or both that donot affect the function of the polypeptides. The additional amino acidsequences can but do not have to assist in purification, detection, orstabilization of the polypeptides.

Further, a skilled artisan understands that polynucleotide andpolypeptide sequences presented herein can vary somewhat, whether as aresult, e.g., of sequencing error or allelic variation or duplication,from the sequence presented while still retaining their essentialnature, that is, higher expression level in tumor and preneoplasticcells relative to normal cells. Further, the polynucleotides of theinvention include conservatively modified variants of the sequencespresented herein, complementary sequences, and splice variants. In viewof the known degeneracy in the genetic code, the proteins orpolypeptides disclosed can also be encoded by a large number of otherpolynucleotide sequences, all of which are within the scope of theinvention. Polynucleotide sequences that are at least 80% identical tothe polynucleotide sequences that encode the polypeptide sequencesdisclosed herein can be used as hybridization probes for codingsequences and are thus within the scope of the present invention. Thepolynucleotides and polypeptides of the invention include, withoutlimitation, polymorphic variants, alleles, mutants, and interspecieshomologs that (1) are expressed at higher level in tumor andpreneoplastic cells, especially in liver, breast, colon and kidney tumorand preneoplastic cells, (2) bind to antibodies raised against thecoding region of the disclosed polypeptides, (3) specifically hybridizeunder stringent or moderately stringent hybridization conditions to apolynucleotide that encodes a polypeptide of the present invention, or(4) are amplified by primers that amplify a polynucleotide that encodesa polypeptide of the present invention.

Exemplary stringent hybridization conditions include 50% formamide,5×SSC and 1% SDS incubated at 42° C., or 5×SSC and 1% SDS incubated at65° C., followed by washing in 0.2×SSC and 0.1% SDS at 65° C. Exemplarymoderately stringent hybridization conditions include 40% formamide, 1MNaCl and 1% SDS incubated at 37° C followed by washing in 1×SSC at 45°C. These conditions are merely exemplary as one skilled in the art isreadily able to discern stringent from moderately stringenthybridization conditions.

Moreover, the sequences of the invention also encompass substitutions,additions and deletions of the sequences presented where the changeaffects one or a few amino acids in the presented polypeptide sequences,without substantial effect upon the activity of the polypeptide, i.e.,differential expression in cancer cells and preneoplastic cells relativeto normal cells.

The present invention will be better understood upon consideration ofthe following non-limiting example.

EXAMPLE

Materials and Methods

Rapid Amplification of cDNA Ends (RACE). Rapid amplification of cDNAends (RACE) was performed in both directions using the SMART cDNAamplification kit (Clontech) from mouse liver tumor polyA RNA. 5′ and 3′RACE were performed using the gene-specific primers, GSP-A[5′-GCATGGCAAGAACAGACTGG-3′] (SEQ ID NO:5) and GSP-B[5′-GGATGAGAAGGGCATCTGGA-3′] (SEQ ID NO:6). 5′ and 3′ RACE products thatwere identified with the corresponding GSP primer were gel extracted andcloned into TOPO-TA vector (Invitrogen). Cloned products were sequencedby Big Dye (ABI) in the McArdle Laboratory Sequencing Facility(University of Wisconsin-Madison).

RNA Analysis. For analysis of murine CRG-L2 mRNA, total RNA wasextracted from liver using guanidine thiocyanate/CsCl as describedpreviously in (Lukas et al., 1999, incorporated by reference in itsentirety). PolyA mRNA was isolated from 250 μg of total RNA usingOligotex mRNA Kit (Qiagen). RT-PCR was performed as described previously(Graveel et al., 2001, incorporated by reference in its entirety) withprimers, RDA-3a [5′-CAACAACCTGGCTTAGAGC-3′] (SEQ ID NO:7) and RDA-3b[5′-GCCATCTGATGCTCTATCC-3′] (SEQ ID NO:8).

For Northern blot hybridization, polyA RNA samples (2 μg) were preparedand electrophoresed as described previously (Lukas et al., 1999). Gelwas soaked in 5 volumes of water for 5 min and then transferredovernight to a GeneScreen (NEN Life Science Products) membrane in10×SSC. Membrane was UV crosslinked twice (120 mJ) and baked in a vacuumfor 2 h at 80° C. Membrane was prehybridized at 42° C. overnight inhybridization solution [50% formamide, 5× Denhardt's solution, 1% SDS,10% dextran sulfate, 1 mg sonicated salmon sperm DNA (boiled), 5×standard saline phosphate with EDTA (SSPE)]. Probes were labeled by nicktranslation (Rigby et al., 1977). A fragment of CRG-L2 (nucleotides188-1243 of SEQ ID NO:1) was released with EcoRI from the pCR-TOPO4vector. ³²P-labeled probe was added to the hybridization buffer andhybridized overnight at 42° C. Blots were washed at RT in 2× SSPE for 30min and at 65° C. for 45 min in 2× SSPE, 2%SDS. Signals were visualizedby autoradiography or phosphoimagery.

For analysis of CRG-L2 in human tissue, RT-PCR was performed for 25cycles with primers hCRGL2a [5′-CATGGCAAGAACAGACTGGG-3′] (SEQ ID NO:9)and hCRGL2b [5′-GCCAGGAAACATCCCAAACTC-3′] (SEQ ID NO:10) and 10 μl ofthe reaction was electrophoresed on a 1% agarose/EtBr gel. The gels weresoaked in 1× TAE for 5 min, denatured for 30 min [1.5M NaCl, 0.5M NaOH],and neutralized for 30 min [1.5M NaCl, 0.5M Tris (pH 7.2), 1 mM EDTA (pH8.0)]. DNA was transferred to a Hybond N membrane (Amersham) with 20×SSPE overnight. The membrane was baked for 30 min at 80° C. in a vacuumoven and UV crosslinked twice (120 mJ). The membrane was prehybridizedat 42° C. for 3 h in hybridization solution [50% formamide, 5%Denhardt's, 3.4× SSPE, 10% dextran sulfate, 5% SDS, 1% sarkosyl, 100 mgsonicated salmon sperm DNA (SSS), 100 mg boiled SSS]. Probes werelabeled by nick translation (CRG-L2 fragment, nucleotides 188-1243 ofSEQ ID NO:1) and added to the hybridization solution. Membranes werehybridized overnight at 42° C. and were washed for 20 min at RT in 2×SSPE, 01% SDS and for 2 h at 65° C. in 0.5× SSPE, 0.2% SDS. Signals werevisualized by autoradiography and phosphoimagery. All primers used inthis study were synthesized at the University of Wisconsin-MadisonBiotechnology Center.

In Situ Hybridization. In situ hybridization was performed as describedpreviously (Micales & Lyons, 2001, incorporated by reference in itsentirety) with the CRG-L2 plasmid 5-2 (containing nucleotides 82-1243 ofSEQ ID NO:1) and AFP plasmid (containing nucleotides 726-1401 of the AFPmRNA) in the plasmid pCR4-TOPO (Invitrogen). Sense and antisense probeswere synthesized using T7 or SP6 with a MAXIscript kit (Ambion) togenerate ³⁵S uridine triphosphate (UTP)-labeled riboprobes. Hybridizedsections were exposed to emulsion (NTB-2; Eastman Kodak) in the dark for2 weeks before developing. After they were developed, the sections werecounterstained with hematoxylin, mounted and viewed under bothlight-field and dark-field illumination.

Multiple Tissue cDNA Panel. The mouse and human tissue cDNA panels(Clontech) were screened following manufacturer's instructions. After 28cycles, 5 μl aliquots were removed at various timepoints. The mousepanel was screened with primers, GSP-970 and GSP- 1241 (see RACE sectionfor primer sequences), and the human panel was screened with primers,hCRGL2-C [5′-AGGGCCCACCAGGGCAGAAG-3′] (SEQ ID NO:11) and hCRGL2-D[5′-ACATGCTTGGCTGCCGAGGG-3′] (SEQ ID NO:12).

Human Tissue. Human tissue and serum was procured from the University ofWisconsin Surgical Pathology department, National Disease ResearchInterchange, and the NCI Cooperative Human Tissue Network. All samplesanalyzed were primary tissues. As required by our IRB protocol, theidentity of the patients was unknown. The excess tissue was frozen aftersurgery and stored at −70° C.

Results

Cloning of CRG-L2 using RapidAmplification of cDNA Ends. Byrepresentational difference analysis, a 282 bp fragment of anuncharacterized mRNA was isolated (Graveel et al., 2001). Using RT-PCRanalysis with primers located in the RDA fragment, this mRNA showedelevated expression in mouse liver tumors as compared to quiescent,regenerating, or newborn livers (FIG. 1 a). The low level of expressionin the regenerating livers suggested the possibility that the increasedexpression was tumor-specific and would not occur in non-tumorigenicproliferative states of human liver, such as cirrhosis or hepatitis. Thecomplete cDNA was obtained via Rapid Amplification of cDNA Ends (RACE).Products from both 5′ and 3′ RACE were subcloned and sequenced.Sequencing the 3′ RACE products revealed three fragments which wereidentical at their 5′ ends due to the fixed location of thegene-specific primer. However, these fragments differed at their 3′ends, with the longer fragments containing, but extending past, thesequence of the shorter fragments. Each fragment contained a polyA tailat its 3′ end, indicating that there are multiple polyadenylation sites.The 5′ RACE products were all identical. By conceptually combining the5′ and 3′ RACE products, three mRNAs were identified that containedalternative 3′UTRs (FIG. 1 b). The putative start codon is at nucleotide25 and the putative stop codon at nucleotide 1344 (see SEQ ID NO:1).Because it was known that this mRNA was upregulated in murine livertumors yet the function was unknown, this novel mRNA was named CancerRelated Gene-Liver 2 (CRG-L2).

To confirm the presence of all three of the murine CRG-L2 mRNAs and todetermine which mRNA is predominantly expressed, a Northern blothybridization was performed using mRNA from quiescent livers and fourindividual liver tumors. A 1 kb fragment of the CRG-L2 open readingframe was used as a probe and four mRNAs were observed (FIG. 1 c). The2.4, 3.0, and 5.5 kb mRNAs (designated as A, B, and C respectively inFIG. 1 c) correspond to the 1967, 2380, and 4365 bp cloned cDNAs. Thesize of the observed mRNAs was longer than the RACE cDNA products due tothe polyA tails. A fourth mRNA (designated as D) of approximately 10 kbwas faintly detected but was not cloned via RACE presumably due to itslength. As expected based on previous RT-PCR results, none of the mRNAswere observed in the quiescent livers. The 5.5 kb mRNA was thepredominant form in the liver tumors and thus the sequence of the 4365nt mRNA has been deposited in Genbank as CRG-L2 (AF548022, SEQ ID NO:1).

To determine the structure of the CRG-L2 gene, the sequence of the mRNAwas aligned to mouse chromosome 9 (31 cM) using the Jackson Laboratoryand Ensembl Mouse Genome browsers (FIG. 1 d). The CRG-L2 gene iscomprised of 10 exons and nine introns that cover a minimum of 59 kb. Anexact measure of the CRG-L2 gene is not yet possible because there aregaps between the contigs that contain the introns between exons 1 and 2and exons 8 and 9.

The amino acid sequence (SEQ ID NO:2) of the 47.5 kDa CRG-L2 protein wasanalyzed by the SMART analysis program (FIG. 2 a) and was found tocontain two collagen domains near the amino terminus (amino acids 29-88and 89-149 of SEQ ID NO:2) and a large olfactomedin domain within the Cterminus (amino acid 189-433 of SEQ ID NO:2). Hydrophobicity analysis ofthe CRG-L2 protein revealed hydrophobic sequences within the firstthirty amino acids of the amino terminus, which represent a leadersequence, suggesting that CRG-L2 is secreted (FIG. 2 b). A serine wasalso present at amino acid 21 which is anticipated to be the cleavagesite of the leader sequence. Regions of high hydrophobicity were alsopresent in the carboxy terminal region, which is anticipated torepresent transmembrane domains.

The human sequence for CRG-L2 was pieced together by using the UCSCHuman Genome Working Draft (available on the world wide web courtesy ofthe Center for Biomolecular Science & Engineering at the University ofCalifornia-Santa Cruz) to align the sequences. The resulting cDNAsequence is presented as SEQ ID NO:3 and the putative amino acidsequence is presented as SEQ ID NO:4. Using the Wilbur-Lipman DNAalignment method the mouse and human open reading frame (ORF) are foundto be 82.4% identical. Using the Blosum62 alignment method the mouse andhuman predicted protein products are found to be 76% identical. Like themurine protein, the human protein contains two putative collagen domainsand one olfactomedin domain, located at amino acids 27-85, 86-145, and177-395 of SEQ ID NO:4, respectively.

CRG-L2 is localized within chromosome 15q21.2 of the human genome and asimilar intron/exon structure is suggested by comparing the mouse cDNAto the human genome. In the human genome data base at NCBI, cloneHs15_(—)10351 (Genbank Accession No. NT_(—)010194), a contig from humanchromosome 15, has areas of significant homology to the mouse cDNAsequences. Because this region of the human genome has not been finishedin NCBI, the UCSC Human Genome Working Draft was used to align thesequences in piecing together the human sequence for CRG-L2. First,exons of the human CRG-L2 gene were identified by aligning the mouseCRG-L2 ORF to the human genome using the NBLAST program. Next, theidentified exons were spliced together and putative introns were excisedto form SEQ ID NO:3. SEQ ID NO:4 shows a predicted polypeptide sequenceencoded by SEQ ID NO:3. The skilled artisan will appreciate thepossibility for some variation in the polynucleotide and polypeptidesequences arising from uncertainty at putative splice sites.

CRG-L2 mRNA is upregulated in human hepatocellular carcinomas. As notedabove, regions of human chromosome 15 are highly similar to mouseCRG-L2. Based on this similarity, primers were designed to detect humanCRG-L2 mRNA. Using these human primers, the level of expression ofCRG-L2 was measured in multiple human hepatocellular carcinomas and innormal livers. A combined method of RT-PCR and Southern blothybridization was used to measure the levels of human CRG-L2. CRG-L2mRNA was amplified by RT-PCR for 25 cycles and the PCR products weretransferred to a nylon membrane that was probed with a fragment of themurine CRG-L2 open reading frame (nucleotides 188-1243 of SEQ ID NO:1).As shown in FIG. 3, CRG-L2 mRNA is essentially undetectable in thenormal liver samples but can be detected in all five hepatocellularcarcinoma samples (middle panel). Extremely high expression is seen inHCC-2, as seen by the shorter exposure of the film (top panel). Accuratequantitation of the starting mRNA samples was verified by analysis ofGAPDH mRNA.

CRG-L2 is upregulated early in liver tumorigenesis. A very importantcharacteristic of a clinical marker for HCC would be early expressionduring liver tumor development. Because it is difficult to obtainsamples corresponding to early states of liver tumors from human cancerpatients, we investigated the timing of expression of CRG-L2 using theDEN-treated mouse model. After a single administration of DEN to 12 dayold mice, basophilic foci are visible by histological staining at 12weeks of age. Sequential development of hyperplastic nodules,hepatocellular adenomas, and hepatocellular carcinomas is observedbetween 12 weeks and 32 weeks of age in male mice (Moore et al., 1981;Vesselinovitch et al., 1985). Therefore, we sacrificed the DEN-treatedmice at 20 and 32 weeks of age. At 20 weeks of age, numerouspreneoplastic lesions were visible throughout the liver and by 32 weeksthe foci had progressed into hepatocellular adenomas/carcinomas (Haniganet al., 1988). Paraformaldehyde fixed sections from 20 and 32 weeklivers were probed with either an antisense (to detect CRG-L2 mRNA) orsense (negative control) CRG-L2 probe.

We began by analysis of the 32 week tumors because our RT-PCR resultsclearly showed that CRG-L2 is upregulated at this stage. Although weexpected to detect CRG-L2 mRNA in the 32 week tumors, in situhybridization can provide additional information that cannot be obtainedby RT-PCR analysis. For example, tumor-specific genes may demonstrate aconstant level of expression throughout a tumor or the expression can belocalized to specific cell types or spatial locations (e.g. theperiphery of the tumor). Using in situ hybridization, we observed thatCRG-L2 mRNA was upregulated in hepatocytes throughout the entire tumor.We note that CRG-L2 was detected in only 69% (311/453) of the tumorsexamined using in situ hybridization but was detected in all seventumors examined by RT-PCR (FIG. 1 a). This could be due to the fact thatonly seven tumors were analyzed in FIG. 1 a or because RT-PCR is moresensitive than in situ hybridization.

To determine if CRG-L2 is upregulated at early stages ofhepatocarcinogenesis, the expression of CRG-L2 was examined in thepreneoplastic foci using in situ hybridization. Interestingly, we foundthat CRG-L2 mRNA can be detected in preneoplastic foci. The pattern ofCRG-L2 expression appears to be consistent throughout the focus with nolocalization within any individual region. We found that CRG-L2 ishighly upregulated in 55% of the foci (220/403) but that there is noobvious histological differences in those foci which do or do notexpress CRG-L2; e.g. CRG-L2 is upregulated in both basophilic andeosinophilic foci and in foci with extensive fat or collagen deposits.AFP was found to be upregulated in 30% of preneoplastic foci (92/304)although the expression pattern was often restricted to various regionsof the focus and not as uniformly distributed as CRG-L2. In otherstudies, AFP has been shown to be expressed in only 23% of 28 week oldDEN-treated B6C3F1 mice (Koen et al., 1983) and 24% of humanhepatocellular carcinomas by immunohistochemistry (Borscheri et al.,2001). In comparison to AFP in these studies, CRG-L2 may be a moresensitive marker for the detection of early HCC.

CRG-L2 displays restricted expression in normal tissues. Acharacteristic of a good clinical marker for HCC is tumor-specificexpression; i.e. low expression in all normal tissues not just in thetissue from which the tumor is derived. Although CRG-L2 mRNA was notdetected in normal mouse liver, it was possible that the mRNA wasexpressed in other normal tissues. The expression of CRG-L2 was examinedin mouse and human tissues using a multiple tissue cDNA panel. Becauseperforming high numbers of PCR cycles can sometimes obscure differentialexpression, aliquots of the PCR products were taken out after variouscycles (30-34). We found that CRG-L2 is primarily expressed in the mousetestis with moderate expression in skeletal muscle (FIG. 4 a). In humantissues, CRG-L2 was expressed primarily in the placenta (FIG. 4 b). Thepattern of CRG-L2 expression, high in tumors, but normally expressed intestis and placenta, resembles expression patterns of genes known ascancer-testis antigens (CT antigen). Cancer-testis antigens are a groupof genes classified by their exclusive expression in the testis andother reproductive tissues and diverse human cancers. The above findingssuggest that CRG-L2 is a potential CT antigen.

CRG-L2 is a CT antigen. The examination of CRG-L2 expression revealedthat CRG-L2 mRNA is expressed at very low levels in all normal tissuesexcept in the mouse testis and human placenta. Therefore, CRG-L2 fallsinto a class of genes designated as cancer-testis antigens (CT antigen).The characteristics of CT antigens are a lack of expression in normaltissues, except reproductive tissues, and high levels of expression in awide range of tumor types. Currently there are more than ten genesidentified that are CT antigens, one of which, PAGE, also shows highexpression levels in the placenta (Brinkman et al., 1998). Most CTantigens map to the X chromosome, but SCP-1 (Tuireci et al., 1998), CT9(Scanlan et al., 2000), and OY-TES-1 (Ono et al., 2001) map to otherchromosomes, as does CRG-L2. CT antigens are intriguing therapeutictargets for immunotherapy because of their limited expression in normaltissues and the fact that the testis and placenta are immune-privilegedsites. However, the biological function and the relationship tomalignancy of most of these genes is unknown (Ono et al., 2001; Scanlanet al., 2002). With regard to CRG-L2, the protein structure indicatesthat CRG-L2 belongs to a family of olfactomedin-related proteins, whichincludes olfactomedin, myocilin/TIGR, noelin-1, and hGC-1.Olfactomedin-related genes have characteristic tissue-restrictedexpression patterns suggesting a specialized function for each protein(Richards et al., 1998; Zhang et al., 2002). Based on tissuelocalization of several olfactomedin family members and the function ofTIGR/myocilin, it is possible that olfactomedin-related proteins play animportant role in protein-protein interactions within the extracellularmatrix (Kulkami et al., 2000). CRG-L2 also contains two collagendomains; proteins that contain collagen domains are also often involvedin the structure of the extracellular matrix.

Our results clearly indicate that expression of CRG-L2 is increased intumors. This increased expression in tumors and restricted pattern ofexpression in normal tissues indicates that CRG-L2 is a tumor-specificantigen. It is thus anticipated that a patient will display animmunogenic response to CRG-L2, making CRG-L2 a marker that can bedetected using blood samples to allow more cost-effective screening of alarger number of high risk patients.

The polynucleotide and polypeptide sequences disclosed herein provide askilled artisan with the ability to assess using conventional methodsthe expression levels of the human CRG-L2 gene in an array of tissuesand more specifically to monitor the expression of the gene in humanliver regions suspected of liver cancer or preneoplastic development ascompared to normal human liver tissue. Likewise, antibodies directed toa portion of the human protein can be produced and used as diagnosticagents for assessing protein levels in various human tissues includingliver tumors. In addition, over-expressed CRG-L2 from liver tumor cellsand preneoplastic liver cells is expected to be secreted into the bloodand the blood level of this protein can be easily monitored by variousmethods known to one of ordinary skill in the art. A patient havingliver cancer and preneoplastic development in the liver may also developan immune response to CRG-L2 and thus an antibody to CRG-L2 may bedetected in the blood of the patient.

The present invention is not intended to be limited to the foregoingexample, but rather to encompass all such variations and modificationsas come within the scope of the appended claims.

REFERENCES

(All references listed below are herein incorporated by reference intheir entirety)

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1. An isolated nucleic acid comprising a polynucleotide selected fromthe group consisting of (1) a first nucleotide sequence that encodes apolypeptide selected from the group consisting of amino acid 22 to aminoacid 439 of SEQ ID NO:2, amino acid 22 to amino acid 400 of SEQ ID NO:4,a sequence that is at least about 68% identical to amino acid 22 toamino acid 439 of SEQ ID NO:2, and a sequence that is at least about 68%identical to amino acid 22 to amino acid 400 of SEQ ID NO:4, (2) asecond nucleotide sequence that is at least 80% identical to the firstnucleotide sequence, and (3) a complement of the first or secondnucleotide sequence.
 2. The isolated nucleic acid of claim 1, whereinthe nucleic acid consists of a polynucleotide selected from the groupconsisting of (1) a first nucleotide sequence that encodes a polypeptideselected from the group consisting of amino acid 22 to amino acid 439 ofSEQ ID NO:2, amino acid 22 to amino acid 400 of SEQ ID NO:4, a sequencethat is at least about 68% identical to amino acid 22 to amino acid 439of SEQ ID NO:2, and a sequence that is at least about 68% identical toamino acid 22 to amino acid 400 of SEQ ID NO:4, (2) a second nucleotidesequence that is at least 80% identical to the first nucleotidesequence, and (3) a complement of the first or second nucleotidesequence.
 3. The isolated nucleic acid of claim 1, wherein the nucleicacid comprises a polynucleotide selected from the group consisting ofnucleotide 88 to nucleotide 1341 of SEQ ID NO:1, nucleotide 64 tonucleotide 1200 of SEQ ID NO:3, and a complement of any of theforegoing.
 4. A genetic construct comprising a polynucleotide of claim 1operably linked a heterologous transcriptional promoter.
 5. A host cellcomprising the genetic construct of claim
 4. 6. The isolated nucleicacid of claim 1, wherein the nucleic acid comprises a polynucleotideselected from the group consisting of (l) a first nucleotide sequencethat encodes a polypeptide selected from the group consisting of SEQ IDNO:2, SEQ ID NO:4, a sequence that is at least about 70% identical toSEQ ID NO:2, and a sequence that is at least about 70% identical to SEQID NO:4, (2) a second nucleotide sequence that is at least 80% identicalto the first nucleotide sequence, and (3) a complement of the first orsecond nucleotide sequence.
 7. The isolated nucleic acid of claim 6,wherein the nucleic acid consists of a polynucleotide selected from thegroup consisting of (1) a first nucleotide sequence that encodes apolypeptide selected from the group consisting of SEQ ID NO:2, SEQ IDNO:4, a sequence that is at least 70% identical to SEQ ID NO:2, and asequence that is at least 70% identical to SEQ ID NO:4, (2) a secondnucleotide sequence that is at least 80% identical to the firstnucleotide sequence, and (3) a complement of the first or secondnucleotide sequence.
 8. The isolated nucleic acid of claim 6, whereinthe nucleic acid comprises a polynucleotide selected from the groupconsisting of nucleotide 25 to nucleotide 1341 of SEQ ID NO:1,nucleotide 1 to nucleotide 1200 of SEQ ID NO:3, and a complement of anyof the foregoing.
 9. A genetic construct comprising a polynucleotide ofclaim 6 operably linked a heterologous transcriptional promoter.
 10. Ahost cell comprising the genetic construct of claim
 9. 11. An isolatedpolypeptide comprising an amino acid sequence selected from amino acid22 to amino acid 439 of SEQ ID NO:2 and a sequence that is at leastabout 68% identical to amino acid 22 to amino acid 439 of SEQ ID NO:2.12. The isolated polypeptide of claim 11 wherein polypeptide consists ofan amino acid sequence selected from amino acid 22 to amino acid 439 ofSEQ ID NO:2 and a sequence that is at least about 68% identical to aminoacid 22 to amino acid 439 of SEQ ID NO:2.
 13. The isolated polypeptideof claim 1 1, wherein the polypeptide comprises amino acid 22 to aminoacid 439 of SEQ ID NO:2.
 14. An isolated polypeptide comprising an aminoacid sequence selected from SEQ ID NO:2 and a sequence that is at leastabout 70% identical to SEQ ID NO:2.
 15. The isolated polypeptide ofclaim 14 wherein the polypeptide consists of an amino acid sequenceselected from SEQ ID NO:2 and a sequence that is at least about 70%identical to SEQ ID NO:2.
 16. The isolated polypeptide of claim 14,wherein the polypeptide comprises SEQ ID NO:2.
 17. An antibody thatspecifically binds to a polypeptide consisting of an amino acid sequenceencoded by the first nucleotide sequence in claim
 1. 18. An antibodythat specifically binds to a polypeptide consisting of an amino acidsequence encoded by the first nucleotide sequence in claim
 6. 19. Amethod for identifying an agent that can modulate the expression of apolynucleotide of claim 1, the method comprising the steps of: exposinga cell that comprises a polynucleotide of claim 1 under the control ofits native promoter; measuring the expression of the polynucleotide inthe cell; and comparing the expression to that in a control cell that isnot exposed to the test agent, wherein a higher or lower than theexpression in the control cell indicates that the agent can modulate theexpression of the polynucleotide.
 20. The method of claim 19, whereinthe cell that comprises a polynucleotide of claim 1 under the control ofits native promoter is a cell selected from the group consisting of aliver cell, a breast cell, a kidney cell and a colon cell.
 21. A methodfor diagnosing a cancer or preneoplastic development in a tissue ororgan of a human or non-human animal, the method comprising the stepsof: measuring the expression of a polynucleotide of claim 1 in cells ofthe tissue or organ obtained from a region suspected of cancer orpreneoplastic development; and comparing the expression of thepolynucleotide to a normal standard wherein a higher than normalexpression indicates cancer or preneoplastic development in the tissueor organ in the suspect region.
 22. The method of claim 21, wherein thetissue or organ is liver.
 23. A method for identifying a human ornon-human animal as a candidate for further screening for cancer orpreneoplastic development in a tissue or organ, the method comprisingthe steps of: determining the level of a polypeptide in a blood orblood-derived sample from the animal wherein the polypeptide comprisesan amino acid sequence encoded by the first nucleotide sequence in claim1; comparing the level to a normal range established by the same animalduring a period that is tumor-free in the tissue or organ, or by aplurality of animals of the same species that are tumor-free in thetissue or organ; and identifying the animal as a candidate for furthercancer screening when the level exceeds the established normal range.24. The method of claim 23, wherein the tissue or organ is liver.
 25. Amethod for identifying a human or non-human animal as a candidate forfurther screening for cancer or preneoplastic development in a tissue ororgan, the method comprising the steps of: determining the level of anantibody to a polypeptide in a blood or blood-derived sample from theanimal wherein the polypeptide comprises an amino acid sequence encodedby the first nucleotide sequence in claim 1; comparing the level to anormal range established by the same animal during a period that istumor-free in the tissue or organ, or by a plurality of animals of thesame species that are tumor-free in the tissue or organ; and identifyingthe animal as a candidate for further cancer screening when the levelexceeds the established normal range.
 26. The method of claim 25,wherein the tissue or organ is liver.
 27. A kit comprising: at least oneof an antibody that specifically binds to a polypeptide consisting of anamino acid sequence encoded by the first nucleotide sequence in claim 1,and a probe that hybridizes to the polynucleotide in claim 1; and atleast one control sample component for which the relative or absoluteamount of the polypeptide or polynucleotide is known.
 28. A kit asclaimed in claim 27 wherein the control sample component is selectedfrom the group consisting of liver cancer cells, preneoplastic livercells, normal liver cells, breast cancer cells, normal breast cells,colon cancer cells, normal colon cells, kidney cancer cells, normalkidney cells, an extract of any of the foregoing cells, a blood samplefrom a human or non-human animal, and a blood-derived sample from ahuman or non-human animal.
 29. A kit as claimed in claim 27, wherein thecontrol sample component is an isolated polypeptide consisting of anamino acid sequence encoded by the first nucleotide sequence in claim 1,or an isolated nucleic acid consisting of the polynucleotide in claim 1.30. A kit comprising: at least one of an antibody that specificallybinds to a polypeptide consisting of an amino acid sequence encoded bythe first nucleotide sequence in claim 6, and a probe that hybridizes tothe polynucleotide in claim 6; and at least one control sample componentfor which the relative or absolute amount of the polypeptide orpolynucleotide is known.
 31. A kit as claimed in claim 30, wherein thecontrol sample component is an isolated polypeptide consisting of anamino acid sequence encoded by the first nucleotide sequence in claim 6,or an isolated nucleic acid consisting of the polynucleotide in claim 6.32. A kit as claimed in claim 31, wherein the isolated polypeptide isselected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4, andthe isolated nucleic acid is selected from the group consisting ofnucleotide 25 to nucleotide 1341 of SEQ ID NO:1 and nucleotide 1 tonucleotide 1200 of SEQ ID NO:3.